Applications for sulindac in preparing anti-lung cancer products

ABSTRACT

This invention discloses uses for sulindac in preparing anti-lung cancer products. This invention provides uses for sulindac in the preparation of products to treat lung cancer. From carrying out cancer drug repositioning for the FDA- and CFDA-approved drug sulindac, experiments for this invention show, based on screening of non-anti-cancer drugs for various cancer cell lines (tissue types) and mutation sites, that sulindac has a new use as an anti-lung cancer medication, thus achieving a new purpose for an old drug.

TECHNICAL FIELD

The present invention relates to the technical field of biology, and itparticularly relates to the application of sulindac in the preparationof anti-lung cancer products.

BACKGROUND

Cancer is the most common as well as the most serious disease thatthreatens human health, and developing effective anti-cancer medicationsis critical to extending patients' lives. Along with the rapiddevelopment of cancer genomics and molecular pharmacology in recentyears, the development of new anti-cancer medications has had relativelygood outcomes. However, since the bottlenecks of large investmentsrequired in the development of new medications and the long-time periodscannot be overcome, as well as the great individual variation in tumorgenetics, many traditional anti-cancer medications are not veryeffective, new medications are expensive, and side effects are not wellunderstood.

In a paper published by the researchers Barabasi A L et. al. in the 2011Nature Reviews Genetics, a molecular network analysis conducted based onGWAS findings and an interactome strategy is expected to reveal new drugtargets and molecular markers for complex diseases, and ultimately toprovide an entirely new understanding of disease pathogenesis andtreatment approaches. Even more noteworthy is that it has beendiscovered in drug repositioning studies that susceptibility geneslocked in by GWAS studies as well as their genes with protein-proteininteraction (PPI) can more easily become indirect targets formedications. This discovery aids in explaining the mechanisms of actionof currently available drugs as well as guiding new drug research. In2014, researchers Okada Y et. al. published a paper in Nature showingthat out of the 101 susceptibility genes for rheumatoid arthritis foundthrough a meta-analysis of GWAS findings, 98 are currently being used asdirect or indirect targets for rheumatoid arthritis medications. Theyalso discovered through drug repositioning research that there aredozens of medications that have been approved for use for otherindications that could be used to treat rheumatoid arthritis.

DISCLOSURE

This research was carried out through integrating cancer gene profilesof the Cancer Gene Census of the Cosmic version 72 cancer histologicaldatabase as well as the protein interactions in the STRING version 10database with Drug Bank Version 4.2, the database of FDA approvedmedications. This obtained candidates for drug repositioning andscreening tests for tumor cell lines were carried out, revealing newanti-cancer drugs. Candidates for tumor suppressing drugs revealed fromthe cancer cell line screening are as follows:

nicardipine, sulindac, estrone, etonogestrel, levonorgestrel,mesalazine, indomethacin, sulfasalazine, balsalazide, irbesartan,ibuprofen, isoprenaline, and pentosan polysulfate.

The primary goal of this invention is to provide a new use for sulindac.

This invention provides uses for sulindac in the preparation of productsto treat lung cancer.

The second goal of this invention is to provide a new use for sulindac.

This invention provides uses for sulindac the preparation of products toinhibit the proliferation of lung cancer cells.

The third goal of this invention is to provide a new use for sulindac.

This invention provides uses for sulindac in the preparation of productsto reduce IC50 values in lung cancer cells.

In the product above, the lung cancer cells mentioned are NCI-H720 orSW900.

In the uses above, the product is a medication or reagent kit.

Uses of sulindac in treating lung cancer are also within scope ofprotection of this invention;

or, uses for sulindac in inhibiting the proliferation of lung cancercells are also within scope of protection of this invention.

Uses of sulindac as a medication for treating lung cancer are alsowithin scope of protection of this invention;

Uses of sulindac as a medication for inhibiting the proliferation oflung cancer cells are also within scope of protection of this invention.

The fourth goal of this invention is to provide a kind of product.

The active ingredient in the product of this invention is sulindac, andit has at least one of the following functions:

1) Treatment of lung cancer;

2) Inhibition of the proliferation of lung cancer cells;

3) Reduction of IC50 values of lung cancer cells.

In the product above, the lung cancer cells mentioned are NCI-H720 orSW900.

In the product above, the product is a medication or reagent kit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a distributed 96-well drug screening culture plate.

FIG. 2 is sulindac sensitivity to NCI-H720 lung cancer cells;EC50=38.5628; IC50=42.7074; R²=0.9950.

FIG. 3 is sulindac sensitivity to SW900 lung cancer cells;EC50=925896.9813; IC50=50.3322; R²=0.9790.

BEST MODE TO CARRY OUT THE INVENTION

Unless otherwise specified, the experimental methods employed in thefollowing examples are standard methods.

Materials, reagents, etc. used in the following examples are allcommercially available unless otherwise specified, and the experimentalmethods employed in the following examples are standard methods.

The analyte drug in the following examples is sulindac, and its chemicalcomposition is as follows:

It is a drug bank product with a catalog number of DB00605.

In the examples below, the product sources for the NCI-H720 and SW900lung cancer cells are as follows:

NCI-H720 ATCC CRL-5838 SW900 ATCC HTB-59

The primary instruments and materials in the examples below are:

DMSO (from Sigma, Cat. No. D4540)

96-well clear bottom cell culture plates (from Corning, Cat. No. 3610)

CellTiter Glo reagent kit (from Promega, Cat. No. G7573)

Doxorubicin positive medication (from MCE, Cat. No. HY-15142)

Fetal Bovine Serum (from Gibco, Cat#10099141)

100 mm petri dish (from Corning, Cat#430167)

RPMI-1640 medium (from Gibco, Cat# A1049101)

DMEM medium (from Gibco, Cat#11995081)

DMEM/F12 medium (from Gibco, Cat#11330057)

EMEM medium (from Gibco, Cat#10370021)

Multidrop 384 cell dispensers (Thermo, Cat#5840150)

EnSpire multi-function plate reader (Perkin Elmer, Cat#2300-001M)

In example 1, CELLTITER-GLO was used to test sulindac against lungcancer

A. Test Plate Preparation

1. Cell Plating

a) The complete medium required for each cell was prepared.

b) Before beginning the experiment, the name of the drug screened forthe cells marked on the 100 mm petri dish was confirmed as well asinformation such as the passage time and number of passages to ensurethe experiment was error-free.

c) Refer to steps d) through i) for procedures for adherent cells; referto steps j) through l) for procedures for suspension cells.

d) When using aseptic technique, a vacuum pump was used to draw the cellculture medium.

e) 2 ml of a sterile PBS solution was used to rinse cell surfaces, and avacuum pump was used to aspirate the PBS waste.

f) A 1 ml 0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution was gentlyadded to the petri dish for cell digestion, and after gently mixingseveral times, the solution covered the cell surfaces. The status ofcell digestion was observed under an inverted microscope, and thetrypsin digestion effect was terminated when cell shedding was about tooccur.

g) 5 ml of pre-warmed 37° C. complete medium was added to the petridishes, and a pipette was used to gently dissociate the cells in orderfor them to shed from the bottom of the petri dish.

h) This cell suspension was transferred to a 15 ml or 50 ml sterilecentrifuge tube and they were centrifuged at 1000 rpm for 5 minutes.

i) A vacuum pump was used to aspirate the medium with aseptic technique.5 ml of pre-warmed 37° C. complete medium was used to resuspend the cellsediment, and it was gently dissociated to mix evenly.

j) A pipette was used to gently dissociate the cells so that they fullyshed from the bottom of the petri dish.

k) This cell suspension was transferred to a 15 ml or 50 ml sterilecentrifuge tube and they were centrifuged at 1000 rpm for 5 minutes.

l) A vacuum pump was used to aspirate the medium with aseptic technique.5 ml of pre-warmed 37° C. complete medium was used to resuspend the cellsediment, and it was gently dissociated to mix evenly.

m) A cell counter was used to count the number of suspended cells andadjust the cell suspension to an appropriate density in the plate tocarry out cell plating experiments.

n) NCI-H720 cells and SW900 cells were handled as described above, andNCI-H720 96-well cell culture plates and SW900 96-well cell cultureplates were obtained, respectively.

The complete medium for NCI-H720 cells was HITES (DMEM: F12 Medium) (alive product), Cat#11330057, with a cells/well density of 16000.

The complete medium for SW900 cells was Leibovitz's L-15 (a liveproduct), Cat#11415064, with a cells/well density of 12000.

2. The Drug Analyte Sulindac was Prepared and Administered (200×FinalConcentration):

1) The Master Plate for the Drug Analyte Sulindac was Prepared

a) DMSO was used to dilute the analyte sulindac to 20 mM for use.

b) 79 μL of the 20 mM analyte prepared in step a) was added to the firstwell in the first row of the dilution plate, and then 54 μL of DMSOsolution was added to the second through ninth wells of the first row.25 μL of solution was aspirated from the first to the second well, andafter mixing well 25 μL of solution was aspirated from the second to thethird well, and this was repeated until the 9th well in order to ensurethat 3.16 dilution of the medication would be carried out one-by-one.

2) Doxorubicin Positive Medication (MCE, Cat. No. HY-15142) Master PlatePreparation

a) DMSO was used to dilute Doxorubicin positive medication to 6 mM foruse.

b) The 6 mM Doxorubicin positive medication solution was added to thedilution plate, and the DMSO solution was incrementally added to theanalyte medication to 1:3.16.

3. Drug Working Board Preparation and Dosing

a) The analyte drug and the positive drug sampling template is as shownin FIG. 1, in which S1208: Positive medication Doxorubicin, DMSO:Positive control well, Cpd 1, 2, 3: Analyte drug, DMSO finalconcentration of 0.5% (DMSO compatibility).

b) 95 μl of cell-specific complete medium was added to the workingplate, each medication to 9 wells. A multi-channel pipettor was used totransfer a series of 5 μl (9 wells) of the diluted solution of theanalyte drug and positive medication Doxorubicin (10×finalconcentration) from the working plate, achieving cell culture media ofvarying concentrations.

c) The NCI-H720 96-well cell plates and the SW900 96-well cell platesprepared in step 1 were removed from the incubator, and 10 μl of thecell culture media (10×final concentration) with varying drugconcentrations as described in b) above was added to the NCI-H72096-well cell culture and the SW900 96-well cell culture plate row-by-rowas shown in FIG. 1. It was placed into a CO₂ incubator at 37° C. for 72hours, obtaining the NCI-H720 96-well drug screening plate and the SW90096-well drug screening plate.

Wells with no medication added acted as controls.

The final concentrations and dosing of the analyte drug, positivemedication Doxorubicin, and control in the 96-well plates were asfollows:

The final concentrations (μM) of the analyte drug in wells 2-10 in FIG.1 are, in order: 100, 31.64557, 10.01442, 3.16912, 1.002886, 0.317369,0.100433, 0.031783, 0.010058;

The final concentration (μM) of the positive medication Doxorubicin inwells 2-10 in FIG. 1 are, in order: 30, 9.493671, 3.004326, 0.950736,0.300866, 0.095211, 0.03013, 0.009535, 0.003017;

In addition, the S1208 well in the 96-well plate (E1-H1 and A12-D12): 10μl of the final concentration 100 μM Doxorubicin solution (solventcontaining 0.5% DMSO complete culture medium solution), DMSO wells(A1-D1, E12-H12, and A11-H11): 10 μl containing 0.5% DMSO completeculture medium solution.

B. CELLTITER-GLO Luminescent Cell Viability Assay System

1. CellTiter-Glo Reagent Preparation

a) The CellTiter-Glo reagent buffer was thawed before using andstabilized to room temperature for use.

b) The CellTiter-Glo reagent frozen substrate was thawed before usingand stabilized to room temperature for use.

c) 100 ml of stabilized CellTiter-Glo buffer was added to the containerwith CellTiter-Glo reagent frozen substrate to adequately resuspend itto form an enzyme/substrate mixture, also referred to as theCellTiter-Glo assay reagent.

d) It was gently mixed and vortexed and inverted multiple times toachieve a uniform solution. In general, the CellTiter-Glo substratereagent will adequately dissolve within 1 minute. It is storedseparately in low-light conditions at −20° C. to await use, and freezingrepeatedly should be avoided.

2. Testing

a) Before testing, the NCI-H720 96-well drug screening plate and theSW900 96-well drug screening plate described in 3 above were stabilizedto room temperature for 20-30 minutes.

b) An inverted microscope was used to observe the conditions of eachgroup of cells in the culture plate and their death patterns, and anyabnormal conditions were noted and retested.

c) 100 μl of CellTiter-Glo reagent (prepared as described in 1 above)was added to all drug screening plates and mixed evenly.

d) It was thoroughly oscillated in a 96-well microplate oscillator for 2minutes to allow the cells to undergo full lysis.

e) It was stored away from light at room temperature for 15 minutesbefore carrying out luminescent signal detection to ensure signalstability.

f) An EnSpire multi-function plate reader was used at 570 nm to read theluminescent signals.

g) Data was processed and analyzed.

The results of the NCI-H720 96-well drug screening plate are shown inFIG. 2.

The results of the SW900 96-well drug screening plate are shown in FIG.3.

The IC50 value was calculated; results are shown in Table 1.

The same method was used to test sulindac's action on the IC50 values ofLOVO (ATCC CCL-229) lung cancer cells; results are shown in Table 1.

It is evident that sulindac has a specific inhibitory effect on theproliferation of lung cancer cells and it can be used as a medicationfor lung cancer treatment.

TABLE 1 IC50 values of various cells under the effect of sulindac CellsIC50 value SW900 50.3322 NCI-H720 42.7074 LOVO 100

INDUSTRIAL APPLICATIONS

From carrying out cancer drug repositioning for the FDA- andCFDA-approved drug sulindac, experiments for this invention show, basedon screening of non-anti-cancer drugs for various cancer cell lines(tissue types) and mutation sites, that sulindac has a new use as ananti-lung cancer medication, thus achieving a new purpose for an olddrug.

1-9. (canceled)
 10. A method of treating lung cancer in a subjectcomprising administering an effective amount of sulindac to the subject.11. The method of claim 10, wherein the lung cancer is atypicalcarcinoid or squamous cell carcinoma.
 12. A method of inhibiting theproliferation of lung cancer cells comprising contacting the cells withan effective amount of sulindac.
 13. The method of claim 12, wherein thelung cancer cells are NCI-H720 cells or SW900 cells.
 14. Apharmaceutical composition for use in treating lung cancer comprisingsulindac.
 15. The pharmaceutical composition of claim 14, wherein thelung cancer is atypical carcinoid or squamous cell carcinoma.
 16. A kitcomprising the pharmaceutical composition of claim 14 and instructionsfor use in treating lung cancer.
 17. A method for treating a lung cancerpatient with atypical carcinoid, the method comprising: (a) screening atumor isolated from the patient having lung cancer for an atypicalcarcinoid phenotype; and (b) treating the patient screened in step (a)and having the atypical carcinoid phenotype by administering aneffective amount of sulindac to the patient.